In 2010, we generated a soluble protein of (i) the VP8 subunit of VP4 of human rotavirus P genotype 8 (Wa strain), P genotype 4 (DS-1 strain) and P genotype 6 (1076 strain);(ii) the Wa VP8-EB NSP4 (amino terminal half of the NSP4) fusion protein;(iii) the P2 (a universal T cell epitope of tetanus toxin)-Wa VP8 fusion protein expressed in E. coli;and (iv) Wa VP8 which is conjugated to polysaccharides of P. suis (generated by Drs. Szu and Li of LDMI, NICHS). The combination of the 3 P genotypes (i.e., P genotypes 4, 6 and 8) constitutes more than 95% of all rotavirus P genotypes detectable worldwide today, which are found in association with G genotypes 1, 2, 3, 4, 5, 8, 9 and 12. Guinea pigs immunized parenterally with a purified Wa VP8, DS-1 VP8 or 1076 VP8 protein developed high levels of neutralizing antibodies to a corresponding homologous virus and moderate to low levels of neutralizing antibodies to heterologous strains. Guinea pigs immunized parenterally with a purified P2-Wa VP8 developed higher levels of neutralizing antibodies to both homologous and heterologous rotavirus strains. Parenteral immunization of guinea pigs with a purified Wa VP8-EB NSP4 fusion protein did not increase levels of neutralizing antibodies. Sublingual (mucosal) immunization of guinea pigs with this protein is underway. Our hope is to use the NSP4 protein (a viral enterotoxin) as an immunostimulating factor to increase the immunogenicity of the recombinant subunit proteins including the VP8. We are in the process of constructing P2-DS-1 VP8, P2-1076 VP8 and P2-Wa VP8-EB NSP4 fusion proteins. In addition, we hope to perform a challenge study of P2-Wa VP8 protein in gnotobiotic piglets.